Project Summary Several genera of maternally inherited symbiotic bacteria affect the reproductive biology of their host species by favoring infected female over male offspring. Wolbachia are the archetypes of this adaptive strategy and exist globally in an estimated 40% of all arthropod species and many filarial nematodes, making them one of the most prevalent bacterial infections on the planet. In a variety of arthropods including Coleoptera, Diptera, and Lepidoptera, as well as arachnids, Wolbachia can selectively kill sons of infected females in a process dubbed male killing. This form of sex-specific lethality enhances the spread of the bacteria by increasing the fitness of transmitting females through reduced competition with their dead brothers for limited resources. It can also spur host evolutionary responses including selection on hosts for male mate choice or Wolbachia density suppression as males become rare in populations experiencing high levels of male killing. Regarding vector control, new population models specify that male killing bacteria can speed up the eradication of target populations when used in conjunction with the sterile insect technique. However, despite decades of research on male killing and its relevance to ecology, evolution, and vector control, the microbial genetic basis remains enigmatic and one of the field's most central challenges to solve. We recently identified a candidate gene, hereafter known as WO Male Killing (wmk) from the prophage WO region of Wolbachia, that can cause male killing. When wmk is transgenically expressed in uninfected D. melanogaster embryos, it kills a significant number of male offspring as embryos. Additionally, cytological defects caused by male killing Wolbachia are enriched in wmk-expressing embryos versus controls. Furthermore, the defects occur around the time that the host dosage compensation complex (DCC) forms, and the DCC has previously been linked to bacterial male killing. As wmk represents a potential breakthrough in the search for a microbial gene that causes male killing, this project will test the central hypothesis that wmk is responsible for male killing, which acts by altering the activity of the host DCC. In Aim 1, we will assess if the developmental and cytological defects caused by transgenic wmk expression are the same as those of male killing Wolbachia. In Aim 2, we will test if wmk- induced male death is dependent upon a functional DCC or interacts with other host pathways. If successful, this research will verify the discovery of the first prophage gene that induces male killing and inform the future use of transgenic or paratransgenic strategies to curb the transmission of vector-borne diseases.